Naringin‐inlaid silk fibroin/hydroxyapatite scaffold enhances human umbilical cord‐derived mesenchymal stem cell‐based bone regeneration

Abstract Objectives Large bone defects are a common, debilitating clinical condition that have substantial global health and economic burden. Bone tissue engineering technology has become one of the most promising approaches for regenerating defective bones. In this study, we fabricated a naringin‐inlaid composite silk fibroin/hydroxyapatite (NG/SF/HAp) scaffold to repair bone defects. Materials and Methods The salt‐leaching technology was used to fabricate the NG/SF/HAp scaffold. The cytocompatibility of the NG/SF/HAp scaffold was assessed using scanning electron microscopy, live/dead cell staining and phalloidin staining. The osteogenic and angiogenic properties were assessed in vitro and in vivo. Results The porous NG/SF/HAp scaffold had a well‐designed biomimetic porous structure with osteoinductive and angiogenic activities. A gene microarray identified 854 differentially expressed genes between human umbilical cord‐derived mesenchymal stem cells (hUCMSCs) cultured on SF‐nHAp scaffolds and cells cultured on NG/SF/HAp scaffolds. The underlying osteoblastic mechanism was investigated using hUCMSCs in vitro. Naringin facilitated hUCMSC ingrowth into the SF/HAp scaffold and promoted osteogenic differentiation. The osteogenic and angiogenic capabilities of cells cultured in the NG/SF/HAp scaffold were superior to those of cells cultured in the SF/HAp scaffold. Conclusions The data indicate the potential of the SF/HAp composite scaffold incorporating naringin for bone regeneration.

Bone tissue engineering technology has emerged as a promising approach for the regeneration of defective bones. [9][10][11][12] Bone tissue engineering includes four elements: seed cells, growth factors, scaffolds and the culture environment. 13 The design of engineered bone grafts requires a balance between biocompatibility and mechanical properties. 14 Polymer-or organic-based scaffolds are easily fabricated into different structures but often do not have the desired compressive modulus. [15][16][17] Ceramic-based scaffolds have a high compressive modulus but low porosity and thus display higher rates of engraftment failure than other types of scaffolds. 18 Therefore, composite scaffolds may enhance the mechanical and biochemical properties of scaffolds and have become promising alternatives to repair and regenerate injured tissues.
Silk fibroin (SF) is a protein derived from the cocoons of the silk worm (Bombyx mori). SF has remarkable mechanical strength, controllable biodegradability, and excellent biocompatibility and is easy to process. 19,20 SF has been extensively used in bone tissue engineering for bone regeneration. 21,22 Composite scaffolds of SF and hydroxyapatite (HAp) fabricated using different approaches promote bone regeneration. 23 However, the pore sizes of the resulting scaffolds are smaller and not easily controllable. The salt-leaching method can precisely control the pore sizes of scaffolds. 24 In a previous study, a novel SF/HAp composite scaffold was constructed by incorporating HAp into an SF-hexafluoroisopropanol (HFIP) solution through salt-leaching. The optimal SF:HAp:salt mass ratio was 1:1:20. 14 Presently, we developed a nanofibrous matrix in threedimensional (3D) porous scaffolds using a salt-leaching technique in conjunction with phase separation, as previously reported. 14 Bone morphogenetic proteins (BMPs) are considered the most potent osteoinductive factors for bone tissue engineering. 25 However, the effective application of growth factors remains challenging because of their short half-lives, susceptibility to degradation, and potential for rapid dilution. 26 Alternative osteogenic inducers to treat bone defects are needed. Naringin is an active flavonoid isolated from citrus fruit extracts. The variety of pharmacological activities of naringin include anti-inflammatory, antiapoptotic, anticancer and antihypertensive activities. 27 We previously demonstrated that naringin abrogates bone loss induced by ovariectomy, 28 sciatic neurectomy 29 and glucocorticoids. 30 More importantly, we first suggested that naringin drives endothelial progenitor cell differentiation and improves angiogenesis during osteoporotic fracture healing. 31,32 Owing to the lack of vascular resources in the inner regions of thick scaffolds, seeded cells do not survive long. 33 Naringin may also solve the problem of insufficient blood vessels in the scaffolds. In vivo and in vitro results suggest that naringin may serve as a replacement to help regenerate damaged bone tissue. 34,35 Mesenchymal stem cells (MSCs) are the most commonly used seed cells in bone tissue engineering technology, including adipose-derived MSCs, peripheral bloodderived MSCs, bone marrow MSCs (BMSCs), and human umbilical cord-derived MSCs (hUCMSCs). hUCMSCs have many advantages over adipose-derived MSCs and BMSCs, including a non-invasive collection procedure, low risk of infection, low immunogenicity and multipotency. 36,37 To the best of our knowledge, this is the first study in which naringin was incorporated into the construction of 3D SF/HAp scaffolds. The fabricated naringin-containing SF/HAp scaffolds were characterized using scanning electron microscopy (SEM) and Fourier transform infrared (FTIR) spectroscopy. In vitro and in vivo osteogenic differentiation assays were performed using alkaline phosphatase (ALP) staining, Alizarin red staining (ARS) and gene expression analyses. Finally, the scaffolds were used for bone defect repair in vivo.

| Synthesis of NG/SF/HAp scaffolds
SF/HAp scaffolds containing different concentrations of naringin (NG/SF/HAp scaffolds) were fabricated using a phase separation technique, as previously described. 14 First, the SF solution was prepared using our previously established protocol. 19 Briefly, small pieces of silk cocoons were degummed by boiling in a 0.02 M Na 2 CO 3 solution for 30 min, rinsed with distilled water to remove sericin, and dried overnight. The resulting fibres were then dissolved in 9.3 M LiBr for 4 h at 60°C and then dialysed with a cellulose dialysis membrane (3500 M w ; Solarbio) against ultrapure water for 72 h to remove the residual LiBr.
The aqueous silk solutions were lyophilized and redissolved in HFIP to yield a 16% w/v solution. Naringin is soluble in organic solvents such as dimethyl sulfoxide (DMSO) and HFIP. Accordingly, different concentrations of naringin (0.03, 0.06, and 0.1%) were blended with the silk solution. The HAp powder was mixed with NaCl particles, and the silk solution was poured over the mixture.
The pore sizes of the granular NaCl ranged from 300 to 400 μm.
The HAp-silk composition was fabricated using a silk:HAp:salt mixture with a mass ratio of 1:1:20. Once the HFIP was completely volatilized, the scaffolds were treated with anhydrous ethanol for 1 d to induce β-sheet formation. The salt was removed by immersing the scaffolds in ultrapure water for 72 h. Scaffolds were cut into 4-mm-diameter cylinders with a thickness of 4 mm and sterilized using cobalt-60 irradiation before the cells were seeded.

| Characterization of naringin/SF/HAp scaffolds
The morphology of the SF/HAp scaffolds was observed using SEM (Carl Zeiss, Oberkochen, Germany). The average size of the SF/HAp scaffolds and the pore size of the scaffolds were calculated using Nano Measure 1.2 image processing software.
The mechanical properties of the different scaffolds were assessed in triplicate after immersion in phosphate-buffered saline (PBS) overnight. The compressive strengths of the scaffold specimens (diameter 5 mm, thickness 5 mm) were measured using the Electro-Force 3230 System (BOSE, Minnetonka, MN, USA) at a constant loading rate of 0.5 mm/min.
The porosity of the composite scaffold was calculated based on the initial n-hexane volume (V1), total volume of the scaffold and nhexane (V2), and residual n-hexane volume (V3) using the following equation 38 : The absorption values at 450 nm for different concentrations of naringin was determined using a microplate fluorometer. Standard curves were drawn based on the absorption values. The 0.1 NG/ SF/HAp scaffolds were immersed in 2 mL of PBS at 37°C and then shaken until the maximum naringin release was achieved. All release tests were performed in duplicate, and the experimental standard error never exceeded 15% of the experimental mean. FTIR spectra of the scaffolds were obtained using an FTIR-7600 spectrometer (Lambda Scientific, Edwardstown, Australia).

| Culture and identification of hUCMSCs
Ethical approval was obtained from the Ethics Committee of Tianjin Hospital (Tianjin, China). hUCMSCs were isolated and cultured as described previously. 39 Briefly, after informed consent was provided by patients, and fresh human umbilical cords were obtained postpartum, as previously described. Umbilical cords were disinfected in 75% ethanol for 1 min, and the umbilical cord vessels were removed using ophthalmic scissors. The mesenchymal tissue (Wharton's jelly) was diced into cubes (approximately 0.5 cm) and centrifuged at 250 × g for 5 min. The mesenchymal tissue was washed with serum- The digested cells were washed twice with PBS and centrifuged at 300 × g for 5 min. The supernatants were discarded, and the cells were washed once in the staining buffer. The washed cells were resuspended in staining buffer at a density of 5 × 10 6 cells/mL. Next, 100 μL of cells were placed in a flow tube, and 5 μL of blocking solution was added and incubated at room temperature for 15 min.
Then, 10 μL of blocking solution, antibody combination I (anti-human CD90 FITC, anti-human CD105 APC, and anti-human CD45 PE-Cy7), antibody combination II (anti-human HLA-DR FITC, anti-human CD73 APC, and anti-human CD34 PE-Cy7) and the isotype control were added and incubated at room temperature for 15 min in the dark. Next, 1 mL of staining buffer was added to these four flow tubes and centrifuged at 300 × g for 5 min. The supernatants were discarded, 500 μL of staining buffer was added and mixed well, and flow cytometry was performed using an FACSAria III Cellsorter (BD Biosciences, San Jose, CA, USA).

| Cellular metabolic activity
Cell adhesion and proliferation on the NG/SF/HAp scaffolds were evaluated quantitatively at 1, 3 and 5 days after seeding. After disinfection, the surfaces of the scaffolds were seeded with hUCMSCs at a density of 1 × 10 6 cells/mL until they reached a saturated state. The seeded scaffolds were placed in a 37°C incubator for 1 h and then transferred to another 24-well plate, followed by addition of 1 mL of culture medium to each well. After incubation for 3 days, all scaffolds were washed twice and fixed with 4% paraformaldehyde (Solarbio) at room temperature for 24 h. The scaffolds were dehydrated and dried with a gradient of alcohol solutions for 20 min. Cell viability was assayed using a live/dead cell kit (Thermo Fisher Scientific) according to the standard protocol to verify the viability of hUCMSCs growing on different NG/SF/HAp scaffolds. After 1, 3 and 5 days of coculture, the culture medium was removed, and the cells were rinsed twice with PBS. Scaffolds were stained with live/dead staining solution (0.5 µL of calcein AM and 2 µL of ethidium homodimer-1 diluted in 1 mL of PBS) for 10 min in the dark in an incubator. Images were acquired using a FluoView 1000 confocal microscope (Olympus, Tokyo, Japan). After 1, 3, and 5 days of cell culture, the hUCMSCs were fixed with a 4% paraformaldehyde solution (Solarbio) for 15 min at room temperature and then permeabilized with 0.5% Triton X-100 (Solarbio) in PBS for 10 min at 4°C. After three washes with PBS, the hUCMSCs were stained with TRITC-conjugated phalloidin (0.5 ml of 5 μg/mL, Solarbio) for 10 min, and the nuclei were counterstained blue with DAPI staining solution (Solarbio). Immunofluorescence was observed using a FluoView 1000 confocal laser scanning microscope (Olympus).
Growth kinetics of hUCMSCs on the scaffolds were evaluated  was added to the wells of the plate and incubated for another 1 h to release calcium-bound Alizarin red S and quantify mineralization.

| Osteogenic differentiation of hUCMSCs
The absorbance at 562 nm was measured using a spectrophotometer and normalized to the protein content. Gene set enrichment analysis (GSEA) was performed using Java GSEA software v2.0.13 (http://www.broad insti tute.org/gsea).

| Quantitative reverse transcription polymerase chain reaction
Total RNA was extracted from tissues and cells using TRIzol reagent (Thermo Fisher Scientific). Total RNA was then reverse-transcribed to cDNA using a ReverTra Ace qPCR RT Kit (Toyobo Co., Ltd., Osaka, Japan). The expression levels of target genes were assessed using quantitative reverse transcription polymerase chain reaction (qRT-PCR) in hUCMSCs. The primers used for the qRT-PCR are listed in Table 1. The relative quantification of the target gene levels compared with the level of the internal control gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was performed using the 2 -ΔΔCt method.

| Western blot analysis
hUCMSCs were extracted and lysed with radioimmunoprecipitation assay lysis buffer. Protein concentration was quantified using a BCA protein assay kit (Solarbio). Whole-cell lysates containing 50 μg protein was separated using 12% sodium dodecyl

| Rabbit femoral distal bone defect
Thirty healthy 2-month-old male New Zealand White rabbits were obtained from the Experimental Animal Centre of Tianjin Hospital.
The mean body weight was 2400 ± 320 g. The rabbits were ran-

| Histological analysis
The distal femurs at 4 weeks post-implantation were harvested and

| Statistical analyses
All results are presented as the mean ±standard deviation and were compared using SPSS software (version 22.0; IBM SPSS, Armonk, NY, USA). Paired or unpaired t tests were applied to compare differences between two groups. One-way or two-way analysis of variance (ANOVA) along with Tukey's multiple comparisons test was used to compare multiple groups. Statistical significance was set at P <.05.  Figure 1B). No statistically significant differences were observed between the pore diameters of the four scaffolds. Figure 1C shows  to approximately 50%, but the weight had not changed significantly ( Figure S2).

| Identification of hUCMSCs
To characterize the cultured hUCMSCs, flow cytometry was used

| Effects of naringin on hUCMSC morphology and proliferation
The effects of naringin on hUCMSC adhesion on scaffolds were assessed using SEM. As shown in Figure 3A,

| hUCMSC biocompatibility on SF/HAp and NG/ SF/HAp scaffolds
The abilities of SF/HAp and NG/SF/HAp to promote osteogenesis were assessed. The ALP activities and calcium contents of the hUC-

MSC cultures with scaffolds incorporated with different wt% nar-
ingin were investigated. The results are presented in Figure 4A. The   Figure 5B and C, respectively. GO analysis of these DEGs is shown in Figure 5D. The main enriched KEGG pathways were the PI3K/Akt, vascular endothelial growth factor (VEGF), hypoxia-inducible factor-1 (HIF-1), p53, oestrogen, FoxO, AMP-activated protein kinase, insulin, mammalian target of rapamycin (mTOR) and thyroid hormone signalling pathways ( Figure 5E).

| RNA sequencing
Using the STRING online database and Cytoscape software, 69 DEGs were filtered into the DEG PPI network, which contained 75 nodes and 165 edges ( Figure 5F). Three significant modules were

| Bone-specific gene expression
After culturing hUCMSCs in SF/HAp and NG/SF/HAp scaffolds for  Figure 6A).
Next, we evaluated the levels of bone formation-related proteins using western blot analysis. The effects of NG/SF/HAp on osteogenesis-related protein and gene expression were consistent ( Figure 6B). The possible involvement of phosphorylated (p)-PI3K and p-Akt was examined to further elucidate the molecular mechanisms underlying the role of NG/SF/HAp in promoting osteogenesis. NG/SF/HAp had no effect on PI3K and Akt expression but strongly promoted PI3K phosphorylation ( Figure 6B), indicating that NG/SF/HAp increased PI3K and Akt activity in hUCMSCs.

| μCT
The gross appearance of the bone defects at

| Histological observation
Newly formed bone and fibrous connective tissues were stained with HE and toluidine blue, respectively, to investigate the remodelled tissue within the bone defect area. HE staining did not show obvious inflammatory reactions in any of the groups ( Figure 8A).
In the control group, only several scattered fibrous tissues were  Figure 8D).
Immunohistochemical staining for CD31 revealed higher expression of CD31 in the group treated with 0.1 NG/SF/HAp scaffolds than in the SF/HAp and control groups ( Figure S5).   Presently, naringin enhanced tube formation in HUVECs. As shown F I G U R E 9 Systemic toxicity to the heart, kidney and liver of the host animals implanted with SF/HAp and 0.1 NG/SF/ HAp at 4 and 8 weeks in our previous studies, naringin enhanced angiogenesis in vivo and in vitro. 31,32 Bone active and continuous remodelling occurs in response to physiological loading. An ideal scaffold material for bone tissue engineering requires balanced rates of scaffold degradation and tissue regeneration. During the initial stages of scaffold implantation, the scaffold should possess sufficient strength and stiffness to support in vivo tissue ingrowth. In the later stages of bone tissue repair, scaffold degradation provides extra space for improving bone tissue regeneration. A major limitation of this study was that we did not perform an expanded follow-up study to identify the degree of matching between scaffold degradation and bone tissue growth.

| CON CLUS IONS
A novel porous composite scaffold was fabricated using naringin, SF and HAp. The SF/HAp scaffold incorporated with naringin exhibited favourable biodegradability, biocompatibility, and osteoinductivity in vitro and in vivo. These results indicate the potential usefulness of NG/SF/HAp for bone defect repair and as a degradable implant for clinical orthopaedics.

ACK N OWLED G EM ENTS
Thank you to everyone who participated in this study.

CO N FLI C T O F I NTE R E S T
None. Lei Sun performed the μCT. All authors contributed to write and review the manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data sets generated and analysed during the current study are available from the corresponding author on reasonable request.